Despite many years of research, a comprehensive understanding of the process of viral capsid assembly at the molecular level has not yet been developed. The long-term goal of this project is to understand the mechanistic details of assembly of icosahedral viruses, which can then be used as the basis for development of antivirals targeted at capsid assembly. We propose to investigate assembly of the dsDNA bacteriophage P22, which provides an excellent model for icosahedral virus assembly. Our specific hypothesis is that viral capsid assembly is driven by multiple specific weak protein:protein interactions of the subunits during assembly. Phage P22 first assembles a procapsid into which dsDNA is packaged. In vitro procapsid-like particles can be assembled simply by mixing together coat and scaffolding proteins in the appropriate conditions. The proposed work combines rigorous thermodynamic analysis of assembly with biochemical and genetic approaches. We propose to first characterize the thermodynamics of P22 procapsid assembly to determine how ionic interactions, and entropic and enthalpic forces are involved in correct assembly of capsids. The role of scaffolding protein in proper assembly will be described, also by determining thermodynamic values for the assembly reaction by using scaffolding protein variants. The controlled of addition of capsid subunits during elongation will be characterized through equilibrium analysis of the association of subunits with partial capsids. Secondly, the sites and nature of the interaction between coat and scaffolding protein will be determined by a combination of molecular biology, phage genetics and biochemical techniques. Lastly, how scaffolding protein is organized within procapsids will be established through techniques using electron microscopy. The research proposed is relevant to public health because thoroughly characterizing capsid assembly will allow the step(s) that are the best targets for anti-viral drugs to be identified. In addition, these studies will highlight the important interactions between capsid subunits, which are required for proper assembly of viruses.